Development of stable phosphohistidine analogues

Article abstract of DOI:10.1021/ja104393t

Protein phosphorylation is one of the most common and extensively studied posttranslational modifications (PTMs). Compared to the O-phosphorylation of Ser, Thr, and Tyr residues, our understanding of histidine phosphorylation is relatively limited, partic

Full text of DOI:10.1021/ja104393t

Published on Web 09/29/2010
Development of Stable Phosphohistidine Analogues
Jung-Min Kee,† Bryeanna Villani,‡ Laura R. Carpenter,‡ and Tom W. Muir*,†
Laboratory of Synthetic Protein Chemistry, The Rockefeller UniVersity, 1230 York AVenue, New York,
New York 10065, United States, and ActiVe Motif, Inc., 1915 Saranac AVenue #2, Lake Placid,
New York 12946-1133, United States
Received May 20, 2010; E-mail: muirt@rockefeller.edu
phorylated in the serum. Thus, amino acid analysis and mass
Abstract: Protein phosphorylation is one of the most common
and extensively studied posttranslational modifications (PTMs).
Compared to the O-phosphorylation of Ser, Thr, and Tyr residues,
our understanding of histidine phosphorylation is relatively limited,
particularly in higher eukaryotes, due to technical difficulties
stemming from the intrinsic instability and isomerism of phos-
phohistidine (pHis). We report the design and synthesis of stable
and nonisomerizable pHis analogues. These pHis analogues
were successfully utilized in solid-phase peptide synthesis and
semi-synthesis of histone H4. Significantly, the first antibody that
specifically recognizes pHis was obtained using the synthetic
peptide as the immunogen.
spectrometry are currently the only options for the detection of
pHis.14
To further complicate matters, pHis has two isomeric forms,
3-phosphohistidine (3-pHis, 2) and 1-phosphohistidine (1-pHis, 3),15
both of which have been found in ViVo.14 While chemical
phosphorylation of proteins and peptides can be selectively carried
out on histidine residues, it is very difficult to obtain the 1-pHis
form regioselectively since 1-pHis isomerizes to the more thermo-
dynamically stable 3-pHis under the reaction conditions.14
We envisioned that nonhydrolyzable and nonisomerizable
analogues of both pHis isomers would provide a solution to the
aforementioned problems associated with this elusive PTM. Such
analogues should be useful in the development of pHis antibodies
and the preparation of semi-synthetic pHis-containing proteins
to further investigate the physiological functions of histidine
phosphorylation.
With these goals in mind, we designed phosphoryltriazolylalanine
(pTza) isomers 4 and 7 (Figure 1b) as stable analogues of 3-pHis
and 1-pHis, respectively. On the basis of molecular modeling studies
(see Supporting Information [SI]), these analogues are expected to
mimic the geometry and electronics of pHis. Importantly, the
hydrolytically labile N-P bond is replaced with a nonhydrolyzable
C-P bond.16 In addition, we designed both 1- and 3-pHis analogues
as tools to differentiate the biological functions of each isomer.
Protein phosphorylation is one of the most common and
extensively studied posttranslational modifications (PTMs).1 Phos-
phorylation and dephosphorylation of proteins control the function
of the target protein, and their misregulation has been linked to
numerous human diseases. Accordingly, protein kinases and phos-
phatases have emerged as important drug targets, boosted by the
prominent success of Gleevec, a tyrosine kinase inhibitor used to
treat leukemia and other malignancies.2,3
While most research on protein phosphorylation has focused on
the O-phosphorylation of Ser, Thr, and Tyr residues, histidine can
also be phosphorylated at its imidazole nitrogens. The role of
histidine phosphorylation is well documented in bacterial two-
component signaling pathways;4 however, proteins with phospho-
histidine (pHis) residues are also found in eukaryotic cells.5,6 In
the case of Physarum polycephalum, for instance, pHis accounts
for 6% of the total phosphoamino acids in its basic nuclear proteins.7
The prevalence of pHis is strikingly high among these proteins,
considering that phosphotyrosine (pTyr) is found in less than 1%
of eukaryotic cellular phosphoproteins.8 We are just beginning to
appreciate that histidine phosphorylation plays an important role
in the signaling processes of higher eukaryotes.9 The modification
has been implicated in several processes in recent years, including
G-protein signaling,10 ion conduction,11 secondary metabolism,12
and chromatin biology.13
Despite these discoveries, the intrinsic chemical properties of
pHis and the dearth of adequate research tools have hindered further
understanding of this PTM. In contrast to the phosphate ester in
O-phosphorylated amino acids, the high-energy phosphoramide in
pHis undergoes facile hydrolysis under acidic conditions (Figure
1a), making its detection and isolation from biological sources
difficult. For example, there are no specific antibodies against pHis-
containing proteins since immunogens with pHis will be dephos-
Figure 1. (a) Structures of 3- and 1-pHis. (b) Design of pHis analogues.
Nitrogen atoms in red (H-bond acceptors) are conserved in the pHis
analogues. Hydrolytically labile N-P bonds (red) in pHis are replaced with
stable C-P bonds (blue). Both analogues can be prepared from the same
building blocks by using different catalysts.
Our pHis analogue design also took synthetic practicality into
consideration since these analogues were to be used in solid-phase
peptide synthesis (SPPS), which generally requires excess amounts
of reagents. Lengthy and inefficient syntheses of pHis analogues
† The Rockefeller University.
‡ Active Motif, Inc.
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Scheme 2. SPPS of Histone H4 Tail Peptides with the pHis
would render the subsequent SPPS impractical. In principle, pTza
isomers 4 and 7 can be readily prepared by metal-catalyzed
cycloaddition reactions between an azidoalanine derivative (5) and
an alkyne (6). Due to the mild reaction conditions and functional
group tolerance, the “click” reaction has found a variety of
applications in bioconjugation and amino acid chemistry.17 Sig-
nificantly, either analogue can be obtained from the same building
blocks simply by employing copper or ruthenium catalysts, allowing
divergent syntheses of both isomeric pHis analogues (Figure 1b).18
To our delight, the Cu-catalyzed cycloaddition between Boc-
azidoalanine (8)19 and diethyl ethynylphosphonate (9)20 provided
the desired pTza derivative 10 in good yield (Scheme 1).21 For the
Ru-catalyzed cycloaddition,18,22 it was necessary to protect the
carboxylic acid functionality as a benzyl ester. Simple debenzylation
after the cycloaddition provided the pTza derivative 12. Both
starting materials 8 and 9 are accessible in gram scale in one or
two steps from commercial materials, and this concise route allowed
preparative-scale synthesis of the protected pTza isomers 10 and
12 to be used in the subsequent SPPS.
Analoguesa
a Inset: control peptides used in dot blot assays.
Scheme 1. Synthesis of Protected pTza Isomers
To determine the specificity of Ab-3pHis, we carried out peptide
dot blot assays with a series of histone H4 tail peptides harboring
the two pTza isomers (13, 14) as well as His (15), pTyr (16), pSer
(17), or pThr (18) (Figure 2a). Gratifyingly, Ab-3pHis only
recognizes 13 but not 14-18, demonstrating no cross-reactivity
toward His or other phosphoamino acid residues.
With building blocks 10 and 12 in hand, the synthesis of
phosphono-peptides containing these amino acids was the next goal.
The N-terminal tail of histone H4 was chosen as our initial synthetic
target. Phosphorylation of histidine residues 18 and 75 in histone
H4 has been correlated with increased DNA synthesis in ViVo, and
both 1-pHis and 3-pHis have been detected.13a-c Significantly,
histone H4 histidine kinase (HHK) activity is upregulated by 200-
fold in human hepatocellular carcinomas compared to normal liver
tissues.13d Peptides incorporating stable pHis analogues could be
used as tools to study histone histidine phosphorylation. Accord-
ingly, peptides 13 and 14 were designed that correspond to the
N-terminal tail of human histone H4 (residues 14-23) and contain
either the stable 1-pHis (14) or 3-pHis (13) analogue in place of
His18. Both peptides were synthesized according to standard Boc-
SPPS methods (Scheme 2).23 Following the cleavage from the resin
with HF and RP-HPLC purification, both peptides were successfully
obtained in multimilligram amounts, demonstrating the compat-
ibility of our pHis analogues 10 and 12 with SPPS.
We reasoned that one of the most important applications of our
pHis analogues would be in the generation of antibodies that can
selectively recognize pHis in phosphoproteins. Therefore, peptide
13 was employed as the immunogen to obtain rabbit polyclonal
antibodies.24 It was hoped that such antibodies would cross-react
with a native 3-pHis moiety in the H4 tail, thereby validating pTza
as a mimetic of pHis. The crude sera obtained from inoculated
animals were screened using peptide dot blot assays and the most
promising serum was purified via affinity depletion using a
nonmodified H4 peptide (15) to afford the polyclonal antibody Ab-
3pHis.
Figure 2. (a) Peptide dot blot using Ab-3pHis. The indicated peptides
were labeled with 5-iodoacetamidofluorescein and spotted on a PVDF
membrane (50 pmol each). To check for equal loading, the fluorescence of
the same membrane was recorded at 520 nm upon excitation at 490 nm.
(b) Western blots of recombinant histone H4 and mutants using Ab-3pHis.
For the loading control, the initial blot with Ab-3pHis was stripped and
reprobed with an R-H4 antibody.
To validate the cross-reactivity of Ab-3pHis toward the phos-
phorylated full-length histone H4, recombinant histone H4 protein
was chemically phosphorylated on histidines in 3-pHis form,
following well-established protocols.25 Western blot analysis clearly
shows that Ab-3pHis selectively recognizes the histidine-phospho-
rylated histone H4, but not the nonphosphorylated counterpart
(Figure 2b). Acid treatment of the phosphorylated histone abolishes
this antibody recognition, showing that phosphorylation is indeed
at histidine. Since histone H4 has two histidines (His18 and His75),
we also tested mutant histones lacking each histidine. Experiments
using these mutants show that Ab-3pHis selectively recognizes the
phosphorylation of His18 residue, as per our design. Notably, Ab-
3pHis was also specific for histidine-phosphorylated histone H4
in the presence of a mammalian cell lysate (Figure S5, SI). To the
best of our knowledge, this constitutes the first example of an
antibody that selectively recognizes a pHis-containing protein.
We next utilized pHis analogue, 10, in the SPPS of an R-thioester
peptide, an essential component in preparing proteins Via native
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chemical ligation.26 Proteins harboring our pHis analogues will be
invaluable in investigating the functional role of this PTM. Peptide
R-thioester 19, corresponding to H4 residues 1-22 and containing
pTza isomer 4, was successfully synthesized following standard
protocols.27
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Acknowledgment. We thank Prof. C. David Allis, Peter Moyle
and Muir lab members for valuable discussions. J.-M. K. is a Damon
Runyon Cancer Research Fellow (DRG 2005-09). This work was
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RC2CA148354) and the Starr Cancer Consortium.
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